Abstract:

Claims:

1. A method of mapping the spatial distribution of icroorganism(s) and/or
biofilm(s) in a wound comprising: contacting a membrane with the wound;
and analyzing the membrane to determine the spatial distribution of
microorganism(s) and/or biofilm(s) in the wound.

2. The method, according to claim 1, wherein the membrane
non-specifically adsorbs biological molecules when contacted with the
wound.

3. The method, according to claim 2, wherein said method comprises at
least one of the following steps: blocking the membrane; contacting the
membrane with one or more dyes; rinsing the membrane; and observing the
membrane to determine the location(s) of biofilm(s) and/or
microorganism(s).

4. The method, according to claim 2, wherein, after the membrane is
contacted with the wound, the membrane is contacted with a plurality of
detection ligand molecules that are specific to certain microbial
class(es), genera, and/or species.

5. The method, according to claim 4, wherein at least one detection
ligand molecule is targeted to a component found in extracellular matrix
of microbial biofilm, a polyanionic bacterial exopolysaccharide,
poly-.beta.-(1-6)-N-acetyl-D-glucosamine, alginic acid, or a component
that is specific to certain microbial classes, genera, and/or species.

6. The method, according to claim 2, wherein the membrane is a nylon
membrane.

7. The method, according to claim 6, wherein the membrane is a positively
charged membrane.

8. The method, according to claim 1, wherein the membrane has attached
thereto ligands that specifically bind with components of biofilm and/or
markers associated with particular microbes.

9. The method, according to claim 8, wherein the ligand is a monoclonal
or polyclonal antibody, complementary polynucleotide, DNA aptamer,
protein aptamer, or phage display.

10. The method, according to claim 8, wherein at least one ligand
molecule is targeted to a component found in the extracellular matrix of
microbial biofilm, a polyanionic bacterial exopolysaccharide,
poly-.beta.-(1-6)-N-acetyl-D-glucosamine, alginic acid, or a component
that is specific to certain microbial classes, genera, and/or species.

11. The method, according to claim 8, wherein after the membrane is
contacted with the wound, the membrane is contacted with one or more
detection ligand molecules to facilitate detection of the microbial
components, and the detection ligand molecules are the same as or
different from the ligand molecules that are attached to the membrane.

12. The method, according to claim 11, wherein at least one detection
ligand molecule contains a reporter selected from the group consisting of
fluorescent, chemiluminescent, chromogenic, magnetic, and electromagnetic
signal.

13. The method, according to claim 1, wherein the method is completed in
5, 10, 15, 20, 25, or 30 minutes or less.

14. A chamber for performing the method of claim

15. A membrane for performing the method of claim 1.

16. A kit comprising a membrane of claim 15 and instructions for the use
of the membrane.

Description:

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional application
Ser. No. 61/172,534, filed Apr. 24, 2009, which is incorporated herein by
reference in its entirety.

[0008] The expenditure on chronic wounds is enormous and a financial toll
worldwide. In 2004, the total cost of DFU rose to $10 billion, including
direct expenses (about 4% of the total personal health spending) and
another $5 billion in indirect expenses (disability, nursing homes,
etc.). The majority of the direct cost of DFU (71-88%) is attributed to
in-hospital stay (length of stay being the most important factor) while
the single contribution of other factors (drugs, investigations, surgery,
orthopedic appliances, visits to foot care specialists, home care) is
comparatively low (>10%) (G. A. Matricali, G. Dereymaeker, E. Muls, M.
Flour, C. Mathieu, Diabetes Metab Res.Rev. 23, 339-347 (2007)). Medicare
reimbursement remains insufficient, with hospital costs exceeding
reimbursement by almost $7500 per patient (Matricali et al., 2007,
ibid.).

[0009] The center of disease control (CDC) estimates that among all
nosocomial infections, biofilm-based infections contribute more than 65%
(C. Potera, Science 283, 1837-+ (1999)) which has lead to an increase in
patients' hospitalization by 2 to 3 days and additional costs of over $1
billion per year (L. K. Archibald and R. P. Gaynes, Infect. Dis. Clin.
North Am. 11, 245-255 (1997)). As mentioned previously, the presence of
microbial biofilm is one of the main factors believed to cause delayed
wound healing (R. Edwards and K. G. Harding, Curr. Opin. Infect. Dis. 17,
91-96 (2004); S. G. Jones, R. Edwards, D. W. Thomas, Int. J. Low Extrem.
Wounds 3, 201-208 (2004); and G. A. James et al., Wound. Repair Regen.
16, 37-44 (2008)). In the U.S. alone, chronic wounds affect over 4
million people with treatment costs of $9 billion per year (K. Izadi and
P. Ganchi, Clin. Plast. Surg. 32, 209-222 (2005)). As a consequence,
chronic wound healing is of significant importance to human health as
well as economic development.

[0010] Unfortunately, there are currently no topographical wound
assessment devices for the detection of wound biofilm or microorganisms.
In addition, there is a need for rapid point-of-care devices for
detecting wound bacteria and/or bacterial biofilm. The conventional
method of diagnosing the presence of microorganisms (bacterial and
fungal) in wounds is technologically complex and time consuming,
involving sampling, culturing, and typing in clinical microbiology labs.
This procedure can cause significant delays in assessing the condition of
the wound and administering appropriate treatment.

[0011] In addition to the delay in administering appropriate treatment to
patients due to the time required analyzing samples, it has been well
documented that biofilm in chronic wounds contain a number of
uncultivable and difficult to culture species (P. G. Bowler and B. J.
Davies, Int. J. Dermatol. 38, 573-578 (1999); C. E. Davies et al., J.
Clin. Microbiol. 42, 3549-3557 (2004); and S. E. Dowd et al., BMC.
Microbiol. 8, 43 (2008)), making the characterization of the wound
microflora and identification of potential pathogens or primary
contributors to pathology difficult. This basic deficiency in diagnosis
often results in ineffective treatment strategies.

[0012] Thus, there is a need for a rapid, simple, inexpensive,
point-of-care assay that would detect and localize bacterial biofilms
and/or microorganism in chronic wounds in order to develop more effective
treatment strategies in wound management.

[0014] Biofilms are a major factor that contribute to poor healing of
wounds. Advantageously, the topographical diagnostic materials and
methods of the subject invention can be used to detect the presence and
location of microbial biofilm, as well as microorganisms, on wounds.

[0015] Rapid, point-of-care detection of microbial biofilm and specific
microorganisms (e.g., pathogenic species) that can be accomplished using
the systems and methods of the invention enable more effective treatment
guidelines/strategies in wound management. Such systems and methods
assist health care workers (e.g., physicians) in assessing a wound for
the presence and/or topographical distribution of microbial biofilm
and/or specific microorganisms (class, genera, species etc.). The
point-of-care biofilm and/or microbial wound assay of the subject
invention facilitates designing personalized treatment strategies, as
well as justifying the use of advanced and/or more costly wound
management technologies that may be appropriate to promote wound healing
on a case-by-case basis.

[0016] Furthermore, the data generated according to the subject invention
can be readily archived. This data can be, for example, digital
photography and the topographical data regarding a patient's wound. The
storage and evaluation of this data facilitates long-term comparative
assessment as well as the ability to track trends with respect to
infection characteristics and treatment efficacy in managing
microorganisms and/or microbial biofilm in the wound.

[0017] In a preferred embodiment, the topographical assay of the subject
invention involves taking an impression of a wound and processing the
impression in order to produce a two dimensional map of the location of
microbial biofilm and/or specific microorganisms on the wound.

[0018] In further preferred embodiments of the subject invention, the
primary targets used to indicate the general presence of microbial
biofilm are components found in the extracellular matrix of microbial
biofilm. There can be, for example, signal molecules, polyanionic
bacterial exopolysaccharides such as
poly-β-(1-6)-N-acetyl-D-glucosamine and alginic acid.

[0019] In addition to detecting and locating biofilm, the use of, for
example, reporter-ligands to specific microbial markers (solely or in
addition to reporter-ligands to general microbial biofilm extracellular
matrix targets) allows the presence of specific microbial classes,
genera, and/or species to be located on the biofilm wound map.

[0020] The detection ligand molecules can be, for example, monoclonal or
polyclonal antibodies, DNA aptamers, protein aptamers, phage display, or
any other macromolecular recognition technology. The reporter molecule
can be fluorescent, chemiluminescent, chromogenic, or any other
appropriate electromagnetic signal.

[0021] The appropriate method(s) of visual assessment and data recording
of the biofilm wound map are correlated with the reporter molecule and
assay membrane used in accordance with techniques well known to those
skilled in the art.

[0022] In a second embodiment, the subject invention provides a very,
simple, easy and quick wound map procedure wherein a high capacity
blotting membrane is applied to a wound and then the membrane is
contacted with dye molecules that selectively stain the biofilm matrix.
In this embodiment the membrane does not contain antibodies (or other
specific binding agents) to the biofilm components. Instead, the membrane
binds, for example, proteins, polysaccharides, and DNA in a non-selective
manner. The polyanionic polysaccharide matrix of the biofilm is then
detected by staining with polycationic dye molecules. The dye may be, for
example, alcian blue or ruthenium red.

[0023] In a third embodiment, for wounds in which a topographical map
assay device would be impractical or unfeasible, a point-of-care bacteria
and bacterial biofilm wound assay using a single sampling platform, such
as a swab, is provided. Such a device would be amenable for assessment of
targets other that outer dermal wounds (i.e. eye, ear, etc).

[0024] By using the assays of the subject invention, health care workers
can assess a wound for the presence and topographical distribution of
microbial biofilm and/or specific microorganisms. This biofilm and/or
microbial wound map aids in debridement strategies as well as in
designing personalized treatment strategies. The results of the
topographical distribution assay can also be used to provide
justification for the use of advanced and/or more costly wound management
technologies that may be appropriate to promote wound healing on a
case-by-case basis.

[0025] The use of this device also facilitates assessment of the effect of
various antimicrobial treatment strategies in managing microorganisms
and/or microbial biofilm in the wound.

BRIEF DESCRIPTION OF DRAWINGS

[0026] The file of this patent contains at least one drawing executed in
color. Copies of this patent with color drawings(s) will be provided by
the Patent and Trademark Office upon request and payment of the necessary
fee.

[0027] FIG. 1 shows a schematic of the topographical wound map assay of
the subject invention. Panel A shows a wound "map" affinity membrane blot
that contains the bound ligand (e.g. antibody) being applied to the wound
surface. The ligand binds the unique marker for the target microorganism
or biofilm (e.g., polysaccharide of biofilm matrix). The blot is then
transferred into a first chamber that contains a solution of the same (or
different) ligand that is labeled with a reporter (e.g. alkaline
phosphatase enzyme). After a short incubation time (˜10 minutes)
the wound map blot is transferred into the second chamber to wash out any
unbound labeled-ligand and, after rinsing, the blot is transferred into
the final chamber that contains the substrate for the enzyme of the
labeled-ligand. After a short period of development (˜5 minutes)
the wound map blot strip is rinsed and the areas of wound surface that
contained the microorganism or biofilm are revealed as uniquely "colored"
areas.

[0028] FIG. 2A-H illustrates blots of the top ("wound bed") or bottom of
porcine skin explants onto HYBOND®-N+ membrane and stained with 5
mg/ml Alcian Blue and washed with PBS. A) Blot of the bottom of an
explant with 3 day mature PAO1 biofilm. B) Blot of the top of an explant
with 3 day mature PAO1 biofilm. C) Second blot of the top of an explant
with 3 day mature PAO1 biofilm after blotting on spot B. D) Blot of the
top of an explant with 3 day mature PAO1 biofilm. E) Blot of the top of
an explant with 1 day immature PAO1 biofilm. F) Blot of the bottom of an
explant with 1 day immature PAO1 biofilm. G). Blot of the top of an
unsterile explant (negative control). H) Blot of the bottom of an
unsterile explant (negative control).

[0029] FIG. 3A-F illustrates blots of the top ("wound bed") or bottom of
porcine explants onto HYBOND®-N+ membrane and stained with 5 mg/ml
Ruthenium Red and washed with PBS. A) Blot of the bottom of an explant
with 3 day mature PAO1 biofilm. B) Blot of the top of an explant with 3
day mature PAO1 biofilm. C) Second blot of the top of an explant with 3
day mature PAO1 biofilm after blotting on spot B. D) Blot of the top of
an explant with 3 day mature PAO1 biofilm. E) Blot of the top of an
explant with 1 day immature PAO1 biofilm. F). Blot of the top of an
unsterile explant (negative control).

[0030]FIG. 4 illustrates an embodiment of a rapid, point of care matrix
metalloproteinase (MMP) detector in accordance with the subject
invention.

[0032] FIG. 6A-D illustrates a wound map of Pseudomonas aeruginosa biofilm
on porcine skin explants. A) Blot of the top of a pig skin explant with
one day immature Pseudomonas aeruginosa PAO1 biofilm. Negative controls
include B) blot of the top of a sterilized porcine skin explant without a
biofilm; C) blot of top of unsterilized porcine skin explant; and D) blot
of bottom of unsterilized skin explant.

[0036] FIG. 10A-B illustrates immunodetection of Pseudomonas aeruginosa
biofilm on porcine explant biofilm with fluorescent antibodies to
polyalginic acid, where the cryosections were incubated with a 1:100
dilution of lmg/mL anti-alginate antibody in blocking buffer for two
hours at 4° C. They were then rinsed with PBS w/0.1% Tween 20 and
incubated for one hour with 1:1000 dilution fluorescent anti-human
antibody, then rinsed again and visualized under a fluorescent
microscope. A) is a dark field fluorescent microscopic image of
cryosection of pig skin explant with mature Pseudomonas aeruginosa
biofilm immunostained with antibody to polyalginic acid antibody. B) is
bright field microscopic image of the same cryosection of A).

[0039] Bacterial colonization, particularly the presence of microbial
biofilm, is one of the primary factors that can cause delayed wound
healing. Also, the increased resistance of biofilm to antimicrobial
treatments, relative to planktonic organisms, has been well documented.
Unfortunately, the need for reassessing the efficacy of current
antimicrobial treatments and to develop new treatment strategies specific
for managing microbial biofilm in wounds, particularly chronic wounds,
has only recently become appreciated.

[0040] The subject invention provides point-of-care methods for assessing
the topographical distribution of microbial biofilm and/or specific
microorganisms. Advantageously, the assays of the subject invention can
be used to identify the location of biofilm and/or microbes on a wound,
as well as to provide information about the chemical and/or biological
characteristics of the biofilm and microbes.

[0041] Microbial biofilm distribution on wounds is a dynamic condition.
Knowing the topographical location within the wound of microbial biofilm
and/or microorganisms enables the health care provider to make informed
decisions on the appropriate treatment strategies to be applied to the
wound in a specific localized manner. The point-of-care topographical
biofilm wound map of the subject invention provides health care workers
(e.g. physicians, nurses, and others) immediate information on the
microbial condition of the wound, thereby assisting and justifying the
choice of treatment methods employed to promote wound healing.

[0042] Furthermore, the technology of the subject invention is amenable to
archiving (e.g. digital photography) of the topographical data in the
patient's care record, thereby facilitating long term comparative
assessment.

[0043] In a preferred embodiment, the topographical assay involves taking
an impression of the wound and processing the impression in order to
produce a two dimensional map of the location of microbial biofilm and/or
specific microorganisms (class, genera, species etc.) on the wound. Thus,
the use of this device can aid in, for example, chronic wound treatment.

[0044] The molecule(s) targeted for detection and/or measurement can be
polysaccharides or glycoproteins that contribute to the formation of
biofilms. The primary targets, used to indicate the general presence of
microbial biofilm, are preferably components found in the extracellular
matrix of microbial biofilm (e.g. polyanionic bacterial
exopolysaccharides such as poly-β-(1-6)-N-acetyl-D-glucosamine,
alginic acid, etc.).

[0045] The use of reporter-ligands to specific microbial markers (solely
or in addition to reporter-ligands to general microbial biofilm
extracellular matrix targets) allows the presence of specific microbial
classes, genera, and/or species to be located on the biofilm wound map.

[0046] The detection ligand molecule(s) can be monoclonal or polyclonal
antibodies, DNA aptamers, protein aptamers, phage display, or any other
macromolecular recognition that currently exists or will exist. The
reporter molecule(s) will be fluorescent, chemiluminescent, chromogenic,
or any other detectable signal.

[0047] In one embodiment, the subject invention can be used to detect
Pseudomonas aeruginosa biofilm on a skin wound using a cationic membrane
that binds anions such as polyalginic acid that make up the majority of
the biofilm exopolymeric matrix. After blotting the membrane onto the
wound with the biofilm, the membrane can be stained with a cationic red
dye molecule that binds to the biofilm matrix. Planktonic P. aeruginosa
bacteria (single cells) on wounds that are blotted with the cationic
membrane and stained do not retain any red dye. Thus, the method is
specific for the biofilm exopolymeric matrix.

[0048] Assays can be developed for naked eye or quantitative assessment
using well-established, relatively inexpensive technical and
non-technical personnel.

[0049] The appropriate methods of visual assessment and data recording of
the biofilm wound map can be correlated with the reporter molecules and
assay membranes used. The physical embodiment of the topographical wound
map microbial biofilm reporter assay device correlates with the optimal
means of assessing the target molecules.

[0050] In a second embodiment, the subject invention provides a very,
simple, easy and quick wound map procedure. In this embodiment the
membrane does not contain antibodies to the biofilm components. Instead,
the membrane binds, for example, proteins. polysaccharides, and DNA in a
non-selective manner. The polyanionic polysaccharide matrix of the
biofilm is then detected by staining with polycationic dye molecules. The
dye may be, for example, alcian blue or ruthenium red.

[0051] In yet another embodiment, biomolecules are bound to a non-specific
membrane but then specific ligands are used to identify target molecules
that have bound to the membrane. The specific ligand, may be, for
example, antibodies, aptamers, or other macromolecular recognition
entities.

[0052] In a third embodiment, for wounds in which a topographical map
assay device would be impractical or unfeasible, a point-of-care bacteria
and bacterial biofilm wound assay using a single sampling platform, such
as a swab, is provided. Such a device is amenable for assessment of
targets other that outer dermal wounds (e.g. eye, ear, etc). FIG. 4
illustrates an example of a rapid, point-of-care sampling platform for
indicating matrix metalloproteinase (MMP) detection. A movable wound swab
15 is provided for application to a wound to retrieve a sample. The swab
15 includes a hollow swab shaft 10 connected to buffer in an upper
reservoir. A snap valve 5 releases the buffer from the upper reservoir
into the hollow swab shaft 10. The buffer preferably contains a
substrate. When the buffer enters the hollow swab shaft 10, it preferably
elutes the wound fluid sample from the swab 15. When the reaction is
complete, preferably after a specified period of time, such as, for
example, 5, 10, or 15 minutes, the swab 15 penetrates beyond a foil seal
separating the swab 15 from an inhibitor 20 to stop the reaction. The
inhibitor 20 is preferably a protease inhibitor.

[0053] In a further embodiment, the subject invention provides a kit for
wound mapping. In one embodiment, the kit comprises a membrane as
described herein and instructions for use of the membrane to map wounds.

[0054] By using the assays of the subject invention, the caregiver is able
to assess the biological activity present in the actual wound bed.
Further, the caregiver is more readily able to see the direct impact of
various treatments on the wound. Advantageously, a picture of the wound
environment serves as justification for applying more advanced wound
management technologies on a case-by-case basis (i.e. advance
personalized medicine).

[0055] The assays of the subject invention are amenable to a number of
readily available technologies for assessment and archiving of the
topographical data. For example, chromogenic-luminescence-, or
fluorescence-based detection methods may be used in conjunction with
digital photography for sensitive, intuitive observation and storage of
patient care records. Finally, in addition to describing the
topographical distribution of biofilm and/or microbes, the system can be
adapted to assess multiple analytes (i.e. protease, etc.), thus providing
a more complete assessment of the wound bed.

[0056] Upon conducting the simple procedures of the subject invention, the
healthcare professional has very important information not only to treat
the condition in an as-needed manner, but also to design and justify
subsequent and related treatments, as required by the majority of
insurance corporations.

[0057] The assays of the subject invention can also be used prior to the
application of therapy to ensure that the recipient site is conducive to
the therapy (e.g. any treatment applied to the site will not be adversely
affected by the presence of biofilm or microbes).

[0058] In an embodiment that is specifically exemplified herein, the
subject invention provides assays that can be used to determine and/or
monitor the status of a wound. The assays are quick and easy-to-use. In
specific embodiments, the assays can be carried out by, for example, a
nurse utilizing either no instrumentation or only minimal
instrumentation. In one embodiment, information about the status of a
wound can be readily, easily and reliably generated in 30 minutes or
less. In a preferred embodiment, the results are obtained in 15 minutes
or less. Most preferably, the results are generated in 10 minutes or
less.

[0059] In a specific embodiment, the assays of the subject invention are
utilized to assess the status of chronic wounds. As used herein,
reference to "chronic wounds" refers to wounds that after 2 weeks are not
healing properly.

[0060] Following are examples which illustrate procedures for practicing
the invention. These examples should not be construed as limiting.

[0062] An impression is taken of the wound using a membrane for obtaining
a specimen of microbial biofilm from wounds (FIG. 1A). The impression can
be used to produce copy blots using an appropriate membrane/device, or it
can be processed directly. The wash chamber consists of separate chambers
containing reaction and wash buffers to process the assay membrane.

EXAMPLE 2

Assay Cassette Method

[0063] In one embodiment, the subject invention provides an assay cassette
method for processing a topographical wound map membrane. An impression
can be taken of the wound using a membrane for obtaining a specimen of
microbial biofilm from wounds (FIG. 1A). The impression can be used to
produce copy blots using appropriate membrane(s)/device or will be
processed directly.

[0064] The assay cassette may contain a fluid reservoir at the base
containing a compressible material to hold the reaction buffer. The assay
cassette may contain an upper dry wicking layer employed to pull fluid
through the assay membrane to facilitate the assay reaction and to "wash"
the assay membrane.

EXAMPLE 3

Assay Method

[0065] FIG. 1 is a schematic of one embodiment of the subject invention.
Panel A shows a wound "map" affinity membrane blot that contains the
bound ligand (e.g. antibody) to the unique marker of the microorganism
(i.e. bacteria, fungi) or biofilm. This membrane is applied to the wound
surface. The ligand (e.g. antibody) that is bonded onto the blot binds
with the unique marker for the target microorganism or biofilm (e.g.,
polysaccharide of biofilm matrix). The blot is then transferred into the
first chamber of a developing block that contains a solution of the same
ligand that is labeled with a reporter (e.g. alkaline phosphatase
enzyme). After a short incubation time (˜10 minutes) the wound map
blot is transferred into the second chamber to wash out any unbound
labeled-ligand and after a minute of rinsing the blot is transferred into
the final third chamber that contains the substrate for the enzyme of the
labeled-ligand.

[0066] After a short period of development (˜5 minutes) the wound
map blot strip is rinsed under running tap water and the areas of wound
surface that contained the microorganism or biofilm are revealed as
uniquely "colored" areas.

EXAMPLE 4

Alternative Assay Format

[0067] In one embodiment, the subject invention provides an assay as
follows: [0068] (1) A high capacity binding membrane is used to
non-specifically adsorb biological molecules (including, for example,
polysaccharides, DNA, proteins and lipids) on a wound. In a preferred
embodiment, the membrane is a High Bond nylon sheet. [0069] (2) The
membrane is then submerged in a blocking agent. The blocking agent may
be, for example, serum albumin or casein. The blocking agent coats any
remaining binding sites on the membrane. [0070] (3) The membrane is then
briefly submerged in (or sprayed with) a concentrated solution comprising
a cationic dye. The membrane may be contacted with the dye(s) for, for
example, 1 to 5 minutes, and preferably for about 2-3 minutes. In
specific embodiment the dyes may be alcian blue and/or ruthenium red.
[0071] (4) The membrane is then rinsed in a solution of salt and dilute
acid, with a small amount of methanol or ethanol. In a specific
embodiment, the salt solution can be around 0.9% sodium chloride, the
acid may be acetic acid (or other acid of similar strength) and the
alcohol can be around 1-2%. [0072] (5) The final step is to dry the
membrane and observe the dye-stained area that corresponds to the area of
the wound bed surface that contains a biofilm.

EXAMPLE 5

Assessment of Biofilm Detection: Polyanionic Exopolysaccharides

[0073] Polyanionic exopolysaccharides found in biofilm exopolymeric matrix
were assessed. Preferably, a membrane having a high positive charge (such
as positively charged nylon or activated papers) is used as the target
capture membrane. In contrast to nitrocellulose membranes and uncharged
membranes that have negative charges or no charges, respectively, high
positively charged membranes are able to tightly bind to the highly
negatively charged polysaccharides and bacterial DNA that make up a
majority of exopolymeric material of biofilm. See Table 1 below. In one
embodiment, Amersham HYBOND®-N+ (GE Healthcare), a cationic nylon
membrane, was chosen as the target capture membrane.

[0076] Explant "wound beds" were inoculated with 10 ul of Log phase
bacterial culture of clinically relevant bacterial species (e.g.,
Pseudomonas aeruginosa, Staphylococcus aureus, etc.). The explants were
then placed on soft 0.5% soft agar media containing appropriate
antibiotics (to which the bacteria in planktonic form are not resistant)
to prevent penetration of bacterial biofilm through the bottom of the
explant. The bacteria were cultured for 3 to 5 days, with daily transfer
to fresh media, to produce mature bacteria biofilm. The explants were
treated overnight in liquid media containing 100 MIC of appropriate
antibiotic to kill remaining planktonic bacteria, gently washed with
sterile PBS, and used as desired (e.g., to assess antimicrobial efficacy
of various treatments on immature and mature bacterial biofilm; as a
pseudo biofilm infected chronic wound). In certain experiments, explants
may then be sonicated in PBS with 5 ul/ml Tween-80 in order to obtain
bacterial suspensions for spread plate analysis to determine relative
CFU/ml. The explant or the sonicant bacterial suspension may also be
assessed using microscopy.

[0077] Unsterilized explants and explants in which PAO1 was grown were
blotted, from both sides of the explant, onto the membrane. The blots
were stained with 5 mg/ml of Alcian Blue (FIG. 2) or Ruthenium Red (FIG.
3) for 1 minute, and washed three times with phosphate buffered saline
(PBS) for 30 minutes each. The results showed that, compared to the
unsterilized skin control, both dyes can detect PAO1 biofilm using this
membrane (FIGS. 2 and 3).

[0078] Due to the high background, alternative wash solutions were tested:
PBS with 5 ul/ml Tween-80; PBS with 0.1% SDS; 40% methanol with 10%
acetic acid. Alginate is the primary polyanionic exopolysaccharide
secreted by PAO1 and is the major component of its biofilm matrix. A
solution of 5 mg/ml Alginic acid (Sigma-Aldrich) was 2-fold serially
diluted and 2 μl of each dilution was dotted onto cationic nylon
membrane, stained with 5 mg/ml of Alcian Blue or Ruthenium Red for 1
minute, and washed three times 30 minutes each with one of the wash
solutions. The result show that PBS alone (FIGS. 2 and 3) or with 0.1%
SDS (FIG. 5A) had high background, PBS with 5 ul/ml Tween-80 had reduced
Ruthenium red but not Alcian blue background (FIG. 5B). and 40% methanol
with 10% acetic acid solution removed most of the background for both
dyes (FIG. 5C). Ruthenium red precipitated during staining of the explant
with biofilm blots causing uneven and discolored staining, particularly
for the 3 day biofilm blots (FIG. 3), which was resolved before staining
the unsterile skin blots (FIG. 3G) as well as the dot blots (FIG. 5).

[0079] All patents, patent applications, provisional applications, and
publications referred to or cited herein are incorporated by reference in
their entirety, including all figures and tables, to the extent they are
not inconsistent with the explicit teachings of this specification.

[0080] It should be understood that the examples and embodiments described
herein are for illustrative purposes only and that various modifications
or changes in light thereof will be suggested to persons skilled in the
art and are to be included within the spirit and purview of this
application.